Eliminating genome-wide and transcriptome-wide off-target mutations by base editor
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ABSTRACT:
PROVIDER: PRJNA690814 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Eliminating genome-wide and transcriptome-wide off-target mutations by base editor [tBE_target_genomic_seq]
| PRJNA692189 | ENA
Project description:Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.
2021-03-12 | GSE164477 | GEO
Project description:Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.
2021-03-12 | GSE164837 | GEO
Project description:Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.
2022-11-07 | PXD029363 | Pride
Project description:Transcriptome wide edits after split-engineered base editor treatment
| PRJNA750724 | ENA
Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms. This SuperSeries is composed of the SubSeries listed below.
2019-03-31 | GSE121668 | GEO
Project description:We report transcriptome wide edits comparison between split-engineered base editors and intact base editors. Our results show that, split-engineered base editors show backgound levels of unique C>U edits when compared to intact base editors.
2021-10-18 | GSE181109 | GEO
Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
2019-03-31 | GSE126502 | GEO
Project description:Determinants of base editor efficacy are position-specific
| PRJEB44304 | ENA